Fibroblast growth factor-2 (FGF-2), also called basic fibroblast growth factor (bFGF), is a cytokine which plays a role in the regulation of cell survival, cell division, angiogenesis, cell differentiation and cell migration. It functions as potent mitogen in vitro and is used for the stimulation and proliferation of a wide variety of cell types including human pluripotent cells, mesenchymal stem cells, bone marrow stromal cells and neural stem cells. FGF-2 comprises several isoforms of different lengths including the most used forms with 146 amino acids (isoform 1 according to UniProt database, http://www.uniprot.org, SEQ ID NO:1) and 155 amino acids (isoform 3, SEQ ID NO:2), respectively (FIG. 1). When manufactured in E. coli, isoform 1 is expressed with an additional initiator methionine at its N-terminus, wherein the methionine is typically removed by proteolytic activities of E. coli. It is known that FGF-2 is highly labile at 37° C. which limits its usage in culturing of cells due to rapid loss of biological activity (Furue M. K. et al., Proc Natl Acad Sci USA 105: 13409-13414 (2008), Levenstein M. E. et al., Stem Cells 24: 568-574 (2006)). Heparin, sulfate ion, a number of polysulfated saccharides and heparin-mimicking polymer covalently conjugated to FGF-2 enhance the stability of FGF-2 against thermal denaturation (Shahrokh Z. et al., in: Formulation and Delivery of Proteins and Peptides by Cleland J. L. and Langer R., ACS Symposium Series, Vol. 567, Chapter 6, pp 85-99 (1994); Nguyen T. H. et al., Nature Chemistry 5: 221-227 (2013)).
FGF-2 contains four cysteine residues of which two are surface-exposed and the other two are buried within the protein. Depending on the numbering scheme, the surface-exposed cysteines are at positions 69 and 87 (SEQ ID NO:1), positions 70 and 88 (SEQ ID NO:1 plus the initiator methionine), or positions 78 and 96 (SEQ ID NO:2). The cysteines could form disulfide bridges which may generate disulfide-linked homodimers of FGF-2 which have been described to dissociate to unfolded monomers via spontaneous thiol-disulfide exchange, resulting in aggregation and precipitation (Shahrokh Z. et al., in: Formulation and Delivery of Proteins and Peptides by Cleland J. L. and Langer R., ACS Symposium Series, Vol. 567, Chapter 6, pp 85-99 (1994)).
Recombinant wild-type FGF-2 can be expressed in E. coli in a soluble, active form. Purification of FGF-2 from cell suspension can be conducted by standard chromatographic steps such as heparin affinity chromatography and ion exchange chromatography. The biological activity can be determined by cell-based assays using FGF-2 sensitive cells which, for example, proliferate in the presence of FGF-2.
WO2003/094835 disclosed FGF-2 variants with enhanced receptor subtype specificity. A mutated FGF-2 protein (based on isoform 3) designated “FGF2(3,5Q)-N111G” with the amino acid exchanges A3Q+S5Q+N111G showed an increase in mitogenicity on FGF receptor-1 and FGF receptor-3IIIc (expressing) cells.
U.S. Pat. No. 6,083,706 disclosed methods of inhibiting the export of a leaderless protein such as FGF-2 from a cell expressing the protein. In this context, mutated FGF-2 proteins (based on isoform 3) with the amino acid exchanges C78S and C96S are disclosed. U.S. Pat. No. 6,083,706 does not teach if these mutations have an effect on protein stability or activity.
EP1444995 disclosed the use of animal or human basic fibroblast growth factor (FGF-2) derived proteins for the preparation of biomaterials or medical devices chosen among endovascular prostheses, such as stents and bypass grafts, or coated endoprostheses, or other kinds of medical prostheses, said FGF-2 derived proteins being chosen among FGF-2 mutants which are unable to interact specifically with the Translokin. The amino acid exchange Q65S is disclosed as it is used in one FGF-2 mutant (based on isoform 3) having the mutation combination of Q65S+E68S+R69V+V71E+S73Y.
WO2013090919 disclosed the amino acid exchange K128N in isoform 3, wherein the FGF-2 K128N mutant exhibits increased thermostability relative to wild-type FGF-2, allowing for methods of culturing human pluripotent stem cells in the presence of lower levels of FGF-2 K128N relative to wild-type FGF-2.
Seno M. et al. (Biochem Biophys Res Commun 151: 701-708 (1988)) disclosed the single mutants C70S and C88S in isoform 1 of FGF-2 wherein the biological activity and heparin binding ability was retained when the serine was substituted for the cysteine residue at either 70 or 88 of the FGF-2 protein. This finding indicates that the cysteines at these positions are not essential for expressing biological activity.
Fox G. M. et al. (J Biol Chem 263: 18452-184528 (1988)) disclosed the double mutant C70S+C88S of the FGF-2 protein (isoform 1). This double mutant and natural sequence bovine and human forms were equally active in all assays.
Heath W. F. et al. (Biochemistry 30: 5608-5615 (1991)) disclosed the double mutant C69A+C87S of the FGF-2 protein (isoform 1). Recombinant human FGF-2 (isoform 1) wild-type protein and the double mutant C69A+C87S, an analogue where two of the four cysteines had been replaced by alanine and serine, were equipotent to standard bovine basic fibroblast growth factor.
Arakawa T. et al. (Biochem Biophys Res Commun 161:335-341 (1989)) disclosed a mutant FGF-2 protein in which all four cysteines were replaced by serine. It exhibited mitogenic activity on NIH 3T3 cells which was indistinguishable from the natural sequence molecule.
Rinas U. et al. (Biotechnology (NY) 10: 435-440 (1992)) disclosed mutated FGF-2 proteins (isoform 1) with the single mutation C70S and the double mutation C70S+C88S. Both the single mutation at position 88 and the double mutation at positions 70 and 88 do not greatly alter the partition of bFGF (FGF-2) into soluble and insoluble cell fractions. Thermal stability experiments at 42° C. and 70° C. revealed that cysteine to serine substitutions did not cause aggregation of the folded protein in vitro. No differences in the biological activity of these mutants compared to wild-type FGF-2 were reported.
Caccia P. et al. (Eur J Biochem. 204: 649-655 (1992)) disclosed derivatives of FGF-2 proteins, isoform 3, with chemically modified sulfhydryl groups. Among these, treatment of FGF-2 with iodoacetic acid led to the isolation of a partially carboxymethylated form (Cm-FGF). Peptide mapping analysis of the modified protein showed that two cysteines (78 and 96) were blocked by a carboxymethyl group. Cm-FGF was more stable than the unmodified molecule as measured by HPLC and SDS/PAGE analysis, and more active than unmodified FGF-2 in stimulating proliferation.
US20120225479 disclosed engineered FGF2 molecules with increased thermostability compared to the wild-type protein. The wild-type FGF-2 lost half of its activity when stored at 37° C. in serum-free culture medium for 2 hours and 90% of its activity after 24 hours. The Q65I+N111G+C96S FGF-2 mutant had a 10-fold improvement in thermostability in repeated cell based assays. The combination of Q65I+N111G+C96S mutations increased the expression level of the protein in human cells compared to the wild-type protein. US20120225479 does not teach how a thermostable FGF-2 polypeptide with an increased biological activity, compared to wild-type FGF-2, can be generated.
As mentioned above, FGF-2 is highly labile at 37° C. which limits its usage for example in culturing of cells due to rapid loss of biological activity. Therefore, there is a need in the art for improved FGF-2 polypeptides.
All references cited herein, including patent applications and publications, are incorporated by reference in their entirety.